DESCRIPTION (Copied from applicant's abstrct): The long-term goal of this investigation is to understand the biochemical mechanisms underlying mitochondrial multienzyme complex deficiencies. The model system chosen for study is maple syrup urine disease (MSUD), a severe inborn error in branched-chain amino acid metabolism that produces mental retardation and often fatal ketoacidosis. The enzyme deficient in MSUD, the mitochondrial branched-chain ct-ketoacid dehydrogenase (BCKD) complex, is organized around a 24-meric transacylase (E2) core, to which other enzyme components comprising a thiamine pyrophosphate (TPP)-dependent decarboxylase (El), a dihydrolipoamide dehydrogenase (E3), a specific kinase and a specific phosphatase are attached through ionic interactions. MSUD is genetically heterogeneous and mutations in four (Ela, E1J3, E2 and E3) of the six genetic loci have been described, however, biochemical mechanisms for these mutations are still poorly understood. Recently, our laboratory has determined the crystal structure of the El a2b2 heterotetramer at 2.7 A resolution. Based on this new structural information, we propose to: 1) characterize the mechanism of oxidative decarboxylation catalyzed by human El, and dissect conformational changes in the El active site induced by phosphorylation, 2) decipher the mechanisms by which MSUD mutations may interfere with hydrophobic-core packing, and subunit interactions in El, and develop strategies to mitigate these putative folding defects, 3) define structural determinants in the binding domain of E2 for interactions with El and E3, and determine the three-dimensional structure of the human BCKD complex. Information derived from these studies should increase our understanding of the structure/function and macromolecular assembly of the BCKD complex and help develop more effective therapies for MSUD.